Abrogation of adenosine A1 receptor signalling improves metabolic regulation in mice by modulating oxidative stress and inflammatory responses
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Adenosine is an important regulator of metabolism; however, the role of the A1 receptor during ageing and obesity is unclear. The aim of this study was to investigate the effects of A1 signalling in modulating metabolic function during ageing.
Age-matched young and aged A 1 (also known as Adora1)-knockout (A 1 −/−) and wild-type (A 1 +/+) mice were used. Metabolic regulation was evaluated by body composition, and glucose and insulin tolerance tests. Isolated islets and islet arterioles were used to detect islet endocrine and vascular function. Oxidative stress and inflammation status were measured in metabolic organs and systemically.
Advanced age was associated with both reduced glucose clearance and insulin sensitivity, as well as increased visceral adipose tissue (VAT) in A 1 +/+ compared with A 1 −/− mice. Islet morphology and insulin content were similar between genotypes, but relative changes in in vitro insulin release following glucose stimulation were reduced in aged A 1 +/+ compared with A 1 −/− mice. Islet arteriolar responses to angiotensin II were stronger in aged A 1 +/+ mice, this being associated with increased NADPH oxidase activity. Ageing resulted in multiple changes in A 1 +/+ compared with A 1 −/− mice, including enhanced NADPH oxidase-derived O2 − formation and NADPH oxidase isoform 2 (Nox2) protein expression in pancreas and VAT; elevated levels of circulating insulin, leptin and proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-12); and accumulation of CD4+ T cells in VAT. This was associated with impaired insulin signalling in VAT from aged A 1 +/+ mice.
These studies emphasise that A1 receptors regulate metabolism and islet endocrine and vascular functions during ageing, including via the modulation of oxidative stress and inflammatory responses, among other things.
KeywordsInsulin sensitivity and resistance Islets Metabolic physiology in vivo Metabolic syndrome Oxidative stress Type 2 diabetes Visceral adipose tissue
- ANG II
Dual-emission x-ray absorptiometry
Intraperitoneal glucose tolerance test
Intraperitoneal insulin tolerance test
NADPH oxidase isoform 2
Visceral adipose tissue
Type 2 diabetes is characterised by beta cell dysfunction and insulin resistance [1, 2, 3] leading to endothelial dysfunction with devastating long-term vascular impairment manifested as numerous complications [4, 5]. The incidence of type 2 diabetes increases with age and obesity, both of which are associated with oxidative stress and chronic inflammation. Mechanistic insights into the pathogenesis of type 2 diabetes and novel therapeutic approaches are urgently needed.
Several clinical and epidemiological studies have demonstrated that coffee consumption, mainly caffeine itself, is associated with a reduced risk of developing type 2 diabetes [6, 7, 8]. Caffeine inhibits the receptor-mediated actions of adenosine , which exerts biological effects via four types of receptors (A1, A2A, A2B and A3) . Adenosine is an important regulator of metabolism; it modulates visceral adipose tissue (VAT) function through A1 receptor-mediated actions in decreasing lipolysis and increasing lipogenesis [11, 12]. Studies utilising gene-modified mice have also suggested that the A1 receptor interacts with insulin and glucagon signalling [13, 14] or secretion . Adenosine was demonstrated to mediate metabolically induced vasodilation in several tissues [16, 17], and we have previously shown that A1 receptor activation modulates in vivo islet blood flow in response to glucose .
These results, all obtained in younger animals, indicate that adenosine, via A1 signalling, could affect glucose homeostasis in multiple ways. However, the role of the A1 receptor in age-related metabolic disorders, which is an independent risk factor of type 2 diabetes [19, 20, 21, 22, 23], has not been clearly studied. We hypothesised that abrogation of A1 receptor signalling attenuates metabolic dysfunction associated with ageing and obesity, by modulating islet function, oxidative stress and inflammatory responses. Indeed, our findings demonstrate this and may have therapeutic implications.
This study was approved by the Institutional Animal Care and Use Committee (IACUC) at Georgetown University, and by equivalent IACUCs in Uppsala and Stockholm, and was performed according to the National Institutes of Health guidelines for the conduct of experiments in animals.
Experiments were conducted on adenosine A 1 receptor gene (also known as Adora1)-deleted (A 1 −/−) and wild-type mice (A 1 +/+) from heterozygous breeding pairs. The strain was developed by Johansson and co-workers  and backcrossed by the Jackson Laboratory (Bar Harbor, ME, USA) to a C57BL/6J background. Both sexes were used, with equal distribution for young (3–5 months) and aged (14–16 months) mice. Mice were housed in temperature-controlled rooms with 12 h light/dark cycles and received a standard rodent chow (4% fat, R36, Lactamin AB, Kimstad, Sweden) and tap water ad libitum.
Food intake and body composition analysis
Food intake was assessed (96 h period) and dual-emission x-ray absorptiometry (DEXA) studies were performed using a Lunar PIXImus densitometer (GE Medical-Lunar, Madison, WI, USA) in isoflurane-anaesthetised (Forene; Abbott Scandinavia AB, Solna, Sweden) animals to determine fat and lean masses, as previously described .
Glucose and insulin tolerance test
Intraperitoneal glucose (IPGTT) and insulin tolerance tests (IPITT) were performed, and the acute effects of pharmacological inhibition of the A1 receptor with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a potent and selective antagonist for the A1 receptor (0.2 mg/kg body weight; Sigma-Aldrich; St Louis, MO, USA) or saline (154 mmol/l NaCl; placebo) were investigated. See Electronic Supplementary Material (ESM) Methods for further details.
Pancreatic islet arterioles: vascular reactivity studies
Single islets with attached arterioles were dissected and perfused, as previously described . Arteriolar responses to angiotensin II (ANG II; 10−6 to 10−12 mol/l; Sigma-Aldrich) alone, or together with apocynin (10−4 mol/l; Sigma-Aldrich) were investigated during high (16.7 mmol/l) and low glucose (2.8 mmol/l). Dose–responses to adenosine (10−4 to 10−11 mol/l; Sigma-Aldrich) were also investigated. See ESM Methods for further details.
Pancreatic islets: insulin release and contents
Mouse pancreatic islets were isolated through collagenase digestion and cultured in groups of 150 islets for 3 days. Insulin release was measured in groups of ten islets from each animal after incubation with low (1.67 mmol/l) and high (16.7 mmol/l) glucose. Insulin content in the incubation media and homogenates were determined using a mouse insulin ELISA kit (Mercodia, Uppsala, Sweden) .
Lucigenin-dependent chemiluminescence of superoxide production
NADPH oxidase-mediated superoxide (O2 −) formation was detected by lucigenin-dependent chemiluminescence assay . Pancreas, liver and VAT were separately homogenised and used for subsequent activity measurement. See ESM Methods for further details.
Metabolic markers and cytokines were analysed in mouse blood samples using MesoScale Discovery Multi Array Technology (MSD, Rockville, MD, USA). See ESM Methods for further details.
Pancreas, liver and VAT obtained from young and aged mice under basal conditions, after pretreatment with DPCPX (i.p. 0.2 mg/kg body weight) or 15 min after stimulation with insulin (i.p. 0.75 U/kg body weight), with and without DPCPX-pretreatment, were homogenised. Tissue extracts were prepared for SDS-PAGE followed by western blotting of NADPH oxidase isoform 2 (Nox2; BD Biosciences, Stockholm, Sweden), and total and phosphorylated Akt (Ser473; Cell Signaling/BioNordika, Stockholm, Sweden). Protein bands were quantified using densitometry and results are reported as relative optical density of the specific proteins.
Flow cytometric analysis
Mouse tissues were incubated in digestion medium followed by erythrocyte lysis and cells were stained with antibodies specific for CD11b, F4/80, CD86, MHC II, CD3, CD4, CD8α and respective isotype controls. Samples were analysed in a Gallios flow cytometer. See ESM Methods for further details.
Expression of adenosine receptors
mRNA levels of adenosine A 1 , A 2A , A 2B and A 3 receptors in whole pancreas, VAT and isolated pancreatic islets together with islet arterioles were determined by quantitative PCR. See ESM Methods for further details.
Histology and insulin staining
Pancreatic tissue was fixed and processed for evaluation of islet morphology, volume and insulin content. See ESM Methods for further details.
Values are presented as means ± SEM. Single comparisons between normally distributed variables were tested for significance using the Student’s paired or unpaired t test, as appropriate. For multiple group comparisons, one-way ANOVA followed by Bonferroni’s post hoc test was used to allow for more than one comparison with the same variable. Statistical significance was defined as p < 0.05.
Glucose and insulin tolerance tests
To validate the effect of A1 receptor inhibition on glucose tolerance and insulin sensitivity, paired crossover measurements were conducted in aged mice. DPCPX significantly improved both glucose (Fig. 2g) and insulin (Fig. 2j) responses in A 1 +/+ mice compared with placebo. These effects of the A1 antagonist were not observed in A 1 −/− mice (Fig. 2h, k). Corresponding AUCs are shown in Fig. 2i, l.
Metabolic hormones in plasma
Insulin release and insulin content in isolated islets
Isolated and perfused islet arterioles
NADPH oxidase-mediated superoxide production and Nox2 level
Cytokines in plasma
Populations of macrophages and T cells in VAT
Akt phosphorylation in VAT
Expression of adenosine receptors
To clarify if the different metabolic phenotypes during ageing and between A 1 +/+ and A 1 −/− mice were attributable to the possible differences of adenosine receptors expression, we used quantitative PCR to determine the expression of all four subtypes of adenosine receptors in pancreas (ESM Fig. 3a–d), islets together with their arterioles (ESM Fig. 3e–h) and in VAT (ESM Fig. 3i–l). The A 1 receptor gene expression was undetectable in A 1 −/− mice. In all tissues, no differences in A 2A , A 2B and A 3 receptor expression between genotypes were observed in either young or aged mice. The same adenosine receptor subtypes were also similarly expressed in young and aged mice of the same genotype.
Our major finding is that abrogation of A1 signalling improves the metabolic profile during ageing. The underlying mechanisms are multifactorial: besides the known direct actions on lipolysis and lipogenesis [11, 12], we found actions on peripheral insulin signalling, presumably via attenuation of NADPH oxidase function, as well as modulation of inflammatory pathways in VAT apparently being involved. Moreover, direct effects of A1 signalling on the islet microvasculature, and insulin release, are also involved.
We observed an age-dependent reduction in lean mass and an accumulation of VAT in wild-type but not in A 1 −/− mice. This is entirely compatible with previous studies showing that endogenous adenosine, through A1 receptor signalling, reduces lipolysis and enhances lipogenesis . We also found that advanced age was associated with elevated blood glucose levels, impaired glucose tolerance and insulin responses or signalling in A 1 +/+ but not in A 1 −/− mice. Pharmacological inhibition of the A1 receptor improved glucose tolerance in aged wild-type mice, but had no effect in A 1 knockouts. Our data indicate that not only acute intervention of A1 receptors will affect the metabolic function, but that A1 receptors are also participating in the metabolic derangement in mice during ageing. Taken together, these findings demonstrate that A1 receptor signalling influences both VAT and glucose regulation during ageing and emphasise the potential therapeutic value of targeting of A1 receptors in type 2 diabetes.
Adenosine is known to affect the endocrine pancreas per se , and previous studies suggested that adenosine receptors can modulate insulin and glucagon secretion . We found advanced age to be associated with an elevation of insulin, glucagon, GLP-1 and leptin in A 1 +/+ mice, but in aged A 1 −/− mice only leptin levels were increased. Thus, the beneficial effect of eliminating A1 receptor signalling was extended to the hormone status. Increased leptin concentration or resistance were suggested as contributing to the inflammatory status in adipose tissue  and have been linked to age-associated disorders including obesity, cardiovascular diseases, the metabolic syndrome and diabetes [32, 33, 34, 35, 36]. Elevated glucagon levels and insulin resistance are generally thought to contribute to the pathophysiology of hyperglycaemia in individuals with type 2 diabetes.
Our islet studies demonstrated that the absence of A1 receptors does not directly affect islet morphology or insulin content. However, ageing was associated with reduced glucose-stimulated insulin release in wild-type mice, but this age-dependent reduction was not observed in A 1 −/− mice. Islet blood flow is normally coupled to islet insulin release [37, 38]. To provide a better possibility to study only the islet afferent arteriole, we used a recently developed technique with isolated and perfused single islets with attached arterioles . Similar to that recently described for renal afferent arterioles , islet arterioles from A 1 −/− mice displayed reduced contractility to ANG II. This was apparent during both normo- and hyperglycaemic conditions, although much more profound during the latter. Since the baseline diameters of the islet arterioles were similar in all the groups, one may speculate that, at physiological ANG II concentrations, aged A 1 +/+ mice would have an increased arteriolar resistance. However, future studies with other techniques are required to confirm this hypothesis.
Oxidative stress, particularly O2 −, has been demonstrated to reduce islet blood flow . ANG II stimulates NADPH oxidase-mediated O2 − formation, which contributes to its pronounced vasoactive properties. Interestingly, reduction of oxidative stress by the NADPH oxidase inhibitor apocynin attenuated ANG II-mediated contraction in A 1 +/+ mice, but had no effect on islet arterioles from A 1 −/− mice. This suggests an important role of the adenosine A1 receptor in modulating O2 − production. This notion has also been described in models of ANG II-induced hypertension in which blood pressure elevation and oxidative stress were markedly attenuated in A 1 gene-deleted mice [39, 41].
Increasing evidence from experimental and clinical studies has demonstrated that oxidative stress and inflammation are key factors that contribute to the progression of metabolic disorders including type 2 diabetes [42, 43]. In the present study we show that ageing is associated with increased NADPH oxidase-derived O2 − formation, together with higher Nox2 levels, in both pancreas and VAT from A 1 +/+ mice. This age-dependent elevation in O2 − formation and oxidative stress was clearly attenuated, or even absent, in gene-deleted animals, which certainly may contribute to their better metabolic phenotype.
Age-related VAT accumulation is associated with a chronic, low-grade inflammation and has been increasingly recognised as an independent risk factor of the pathogenesis of type 2 diabetes [44, 45, 46]. Almost all immune cell subsets are present in VAT. Their functions are still under discussion, although it is generally accepted that total T cell and macrophage populations are increased and contribute to the meta-inflammation evident in obesity. We did not discern differences in total macrophages among groups, but observed a significant enhancement of the CD86+ macrophage population in aged A 1 −/− mice, indicating elevated antigen presentation capacity. Interestingly, another recent study reported a lipolysis-related macrophage infiltration in adipose tissue without presenting pro-inflammatory characters . As A 1 −/− mice have elevated lipolysis and remain lean, this enhancement of CD86+ macrophages may be due to a response to the continuous release of non-esterified fatty acids. As young mice have much lower lipolysis levels, no differences (e.g. in fat mass) were evident between genotypes at an early age. However, ageing resulted in a significant increase of total T cells and CD4+ T cells in VAT in A 1 +/+ mice, while this was not the case in A 1 −/− mice. In addition, levels of circulating proinflammatory cytokines (including TNF-α, IL-1β, IL-6 and IL-12) were increased in aged A 1 +/+ but not in A 1 −/− mice. These findings indicate that a sustained inflammation occurs during ageing and obesity, and may lead to subsequent metabolic disorders, as has been previously suggested [48, 49]. Moreover, accumulation of CD4+ T cells in VAT may be a key contributor to this systemic inflammation and can be modulated via A1 receptor signalling. Further investigations on the immunomodulation effects of the A1 receptor during ageing and the metabolic syndrome are needed and will provide additional insights in developing therapeutic strategies for type 2 diabetes. It seems likely that decreased VAT accumulation and inflammation may contribute to the better metabolic regulation in the A 1 −/− mice during ageing, including better insulin sensitivity. Indeed, our data show significantly better insulin signalling in aged A 1 −/− mice, as demonstrated by increased phosphorylation of Akt kinase in VAT upon insulin stimulation. This insulin signalling pathway was markedly reduced in aged-matched wild-type mice and may contribute to their impaired glucose clearance function.
Besides the A1 receptor, also adenosine receptor A2A and A2B play a role in modulating glucose homeostasis and obesity [50, 51]. We did not reveal any significant differences regarding the A2 and A3 receptor expression among the groups, suggesting no compensatory changes following A 1 receptor deletion. Thus, we believe the improved metabolic phenotypes in the A 1 −/− mice in our study were due to the abrogation of A1 signalling and our findings further suggest a pivotal role of the A1 receptor in modulating especially the function of VAT, which may affect the metabolic homeostasis.
We thank E. Lindgren (Karolinska Institutet) and M. Quach (Uppsala University) for technical assistance and helpful discussions.
This work was supported by grants from the Swedish Research Council (521-2011-2639 to MC and 521-2011-3777 to LJ), Swedish Heart and Lung Foundation (20140448, 20110589), Jeanssons Foundation (JS2013-00064), Swedish Diabetes Foundation, an EXODIAb grant, Swedish Society of Medical Research (SSMF), the Wenner-Gren Foundation, the Family Ernfors Fund, the Swedish Society of Medicine and Novo Nordisk Foundation Excellence Project.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
MC, LJ, BBF and TY designed the study and wrote the manuscript. All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, and revised the article critically for important intellectual content. All authors approved the final version of the manuscript to be published. MC is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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